Incubate sample for 5 min at RT to permit dissociation of nucleoprotein complex.

In hood, add 200 µl chloroform to each tube.

Vortex vigorously for 15 s.

Incubate for 3 min at RT.

Centrifuge at 12,000 x g for 15 min at 4°C.

RNA precipitation:

Following centrifugation, sample should have separated into 3 phases:

top: clear aqueous (~50%) - contains RNA

middle: interphase

bottom: red phenol-chloroform

Carefully transfer clear aqueous phase to new tube using 200 µl pipette tips. Leave ~100 µl of aqueous phase in old tube to be sure NOT to contaminate with interphase. Should have about 400-600 µl aqueous phase in new tube, discard rest of solution in Trizol/chloroform waste.

Add 500 µl 100% ethanol to each tube. Mix well by inverting tubes several times.

Transfer 700 µl of sample, including any precipitate, into spin column placed in collection tube. Both tubes from above are combined onto 1 spin column.

Quantify RNA concentration using NanoDrop. Yield should be ~0.5-1 µg RNA (for 1 ml of gel at 105 cells/ml and 7 days in culture). Store RNA at -80°C.

References

Modified from Fan Yang Lab and RNeasy Mini Kit Protocol

Comment

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data including images for the troubleshooting.

You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.